Anti ssadh

Cells were harvested, washed with PBS and cell lysis was performed in 50 mM Tris–HCl pH 7.4 buffer containing 1% Triton X-100, 150 mM NaCl, 0.5 mM EGTA, 0.5 mM EDTA and anti-protease cocktail (Complete Protease inhibitor Cocktail Tablets, Roche, France). Protein extracts (30 μg) were separated by SDS-PAGE and transferred to Hybond-C Extra nitrocellulose membranes (GE Healthcare, USA). The following antibodies were used for immunoblotting: anti-Nanog (Cell Signaling, 1:1000), anti-p21 (Santa Cruz Biotechnology, 1:200), anti-Actin (Millipore Chemicon, 1:10,000), anti-SSAR (Novus biological, 1:2000), anti-ABAT1 (GABA-T) (Sigma Aldrich, 1:2500), anti-GAD65 (Abcam, 1:500), anti-GAD67 (Abcam, 1:500), anti-HOT (Sigma, 1:2000), anti-GLUD1 (Sigma, 1:2000), and anti-SSADH (Novus Biological, 1:2000). The secondary antibodies were anti-mouse IgG (Santa Cruz Biotechnology, 1:10,000), anti-rabbit IgG (GE Healthcare, 1:10,000) and anti-goat IgG (Santa Cruz Biotechnology, 1:10,000). Signal detection was performed with the ECL + chemiluminescence detection system (PerkinElmer, France). Densitometric analysis was achieved using ImageJ software.

Morphologic examination was performed on Hematoxylin- and Eosin-stained sections (3–4 μm). Immunolabeling was performed using an automated system (Autostainer Dako, Glostrup Denmark) with the following primary antibodies: anti-Ki67 (MIB-1, Dako, prediluted), anti-Olig2 (goat polyclonal, R&D Systems, 1:500) and anti-GFAP (Dako, 1:4000). Deparaffinization, rehydration and antigen retrieval were performed using the pretreatment module PTlink (Dako). SSADH immunostaining was achieved following overnight incubation with anti-SSADH (Novus Biological, 1:100) at 4 °C. Immunostaining was scored by a pathologist (FBV).